mir-30 mirna family regulates xenopus pronephros ...the mir-30 mirna family regulates xenopus...

3927RESEARCH ARTICLE

INTRODUCTIONThe kidney is essential to clear waste products, to balance theconcentration of body fluids and electrolytes and to reabsorb smallmolecules, such as amino acids, ions and water, to maintain bloodhomeostasis. It develops from the intermediate mesoderm viainductive processes that form three different and increasingly morecomplex kidney types: the pro-, meso- and metanephros (Saxén,1987; Vize et al., 2003). The metanephros is the adult kidney ofhigher vertebrates, whereas the mesonephros is the adult kidney ofamphibians and fish. The pronephros is of particular importance inaquatic animals during larval stages in order to maintain waterhomeostasis. The development of the three kidney forms isinterconnected and both the meso- and metanephric kidney rely onthe formation of the pronephros (Bouchard et al., 2002; Carroll andMcMahon, 2003; Jones 2003; Dressler, 2006). Moreover,transcription factors, as well as markers of terminal differentiation,have a similar expression pattern and function during kidneydevelopment in human, mouse, zebrafish and Xenopus (Zhou andVize, 2004; Drummond, 2005; Raciti et al., 2008).

MicroRNAs (miRNAs) are a class of ~22 nt, non-codingmolecules expressed in multicellular organisms (Kloosterman andPlasterk, 2006; Bushati and Cohen, 2007; Stefani and Slack, 2008).The principal function of miRNAs is to regulate protein expressionand mRNA stability by binding to complementary nucleotidesequences in the 3�UTRs. miRNAs are initially transcribed by RNApolymerase II as part of a much longer primary transcript (pri-miRNA) with a CAP structure and poly(A) tail. In the nucleus, thepri-miRNA is processed to a ~60 nt hairpin precursor miRNA (pre-miRNA) by the microprocessor complex, consisting of Drosha, an

RNase III type endonuclease, and Dgcr8 (DiGeorge critical region8)/Pasha, a double-stranded-RNA-binding protein. Once cleaved,pre-miRNAs are transported from the nucleus to the cytoplasm,where another RNase III enzyme, Dicer, cleaves pre-miRNA intothe mature ~22 nt duplex miRNA. The mature miRNAs are thenloaded into the RNA-induced silencing complex (RISC), whichregulates binding of miRNAs to the 3�UTR of target mRNAs andinduces translational inhibition or degradation. Target specificity isdetermined by the seed sequence (nucleotides 2 to 8 from the 5� endof an miRNA) and is further strengthened by base pairing offlanking nucleotides (Brennecke et al., 2005; Bartel, 2009). Theexpression of miRNA and of its targets often show an inverserelationship (Farh et al., 2005; Stark et al., 2005). For example,expression levels of predicted targets for miR-1 are high inmyoblasts, but are strongly reduced upon differentiation intomyotubes, when expression of miR-1 is initiated (Chen et al., 2006).

The functions of several individual miRNAs have beencharacterized in detail, such as how lin-4 and let-7 controldevelopmental timing in C. elegans and Drosophila, respectively(Lee et al., 1993; Wightman et al., 1993; Reinhart et al., 2000).However, many miRNAs belong to families with nearly identicalseed sequences that can compensate for each other and make loss-of-function analyses difficult (Abbott et al., 2005; Stefani and Slack,2008). For example, the zebrafish miR-430 family consists of fivemembers that together control the transition from maternal tozygotic gene transcription (Chen, P. Y. et al., 2005; Giraldez et al.,2005; Giraldez et al., 2006). One approach to address the generalrole of miRNAs in development is to interfere with miRNAbiogenesis. Mice lacking either Dicer (Dicer1) or Dgcr8 protein areembryonically lethal owing to defects in germ layer patterning(Bernstein et al., 2003; Wang et al., 2007). But tissue-specificconditional mutants have identified multiple roles for miRNAsduring organogenesis (Stefani and Slack, 2008). In zebrafish,embryos lacking Dicer protein exhibit defects in the degradation ofmaternal transcripts as well as abnormal brain, somite and heartmorphogenesis (Wienholds et al., 2003; Giraldez et al., 2005;

The miR-30 miRNA family regulates Xenopus pronephrosdevelopment and targets the transcription factor Xlim1/Lhx1Raman Agrawal1, Uyen Tran1 and Oliver Wessely1,2,*

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They areinvolved in diverse biological processes, such as development, differentiation, cell proliferation and apoptosis. To study the role ofmiRNAs during pronephric kidney development of Xenopus, global miRNA biogenesis was eliminated by knockdown of two keycomponents: Dicer and Dgcr8. These embryos developed a range of kidney defects, including edema formation, delayed renalepithelial differentiation and abnormal patterning. To identify a causative miRNA, mouse and frog kidneys were screened forputative candidates. Among these, the miR-30 family showed the most prominent kidney-restricted expression. Moreover,knockdown of miR-30a-5p phenocopied most of the pronephric defects observed upon global inhibition of miRNA biogenesis.Molecular analyses revealed that miR-30 regulates the LIM-class homeobox factor Xlim1/Lhx1, a major transcriptional regulator ofkidney development. miR-30 targeted Xlim1/Lhx1 via two previously unrecognized binding sites in its 3�UTR and thereby restrictedits activity. During kidney development, Xlim1/Lhx1 is required in the early stages, but is downregulated subsequently. However, inthe absence of miR-30 activity, Xlim1/Lhx1 is maintained at high levels and, therefore, may contribute to the delayed terminaldifferentiation of the amphibian pronephros.

KEY WORDS: Dgcr8, Dicer, Lhx1 (Lim1; Xlim1), Kidney, microRNA, Pronephros, Xenopus, Mouse

Development 136, 3927-3936 (2009) doi:10.1242/dev.037432

1Department of Cell Biology and Anatomy, and 2Department of Genetics, LSUHealth Sciences Center, MEB 6A12, 1901 Perdido Street, New Orleans, LA 70112,USA.

*Author for correspondence ([email protected])

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Giraldez et al., 2006). However, little information is availableregarding the role of miRNAs in kidney development. Expressionanalyses have identified several miRNAs that are expressed in thekidney (Sun et al., 2004; Naraba and Iwai, 2005; Kato et al., 2007)and conditional alleles eliminating Dicer from mouse podocyteshave demonstrated that miRNAs are required for the maintenanceof functional glomeruli (Harvey et al., 2008; Ho et al., 2008; Shi etal., 2008).

Here we extend the study of miRNAs to the pronephric kidney ofXenopus laevis. Using antisense morpholino oligomers againstDicer or Dgcr8 to inhibit miRNA biogenesis, we show that miRNAsare required for multiple facets of pronephros development,including patterning and terminal differentiation. We identify themiR-30 family as an essential player in this process. Knockdown ofone family member, miR-30a-5p, phenocopies nearly all of thedefects caused by the loss of Dicer or Dgcr8. Finally, we identifiedthe transcription factor Xlim1/Lhx1 as an important target of miR-30 activity. This transcription factor is essential for kidneydevelopment in mouse and frog (Shawlot and Behringer, 1995;Carroll and Vize, 1999; Kobayashi et al., 2005) and we nowdemonstrate that miR-30 is required for its correct spatiotemporalexpression during pronephros development.

MATERIALS AND METHODSEmbryo manipulationsXenopus embryos obtained by in vitro fertilization were maintained in 0.1�modified Barth medium (Sive et al., 2000) and staged according toNieuwkoop and Faber (Nieuwkoop and Faber, 1994). Antisense morpholinooligomers (MOs) were obtained from GeneTools. The sequences of theantisense MOs used in this study were: Dicer-MO, 5�-TGCAG -GGCTTTCATAAATCCAGTGA-3�; Dgcr8-MO, 5�-GGGCTACTT -CCTCACACTCCTCCAT-3�; miR-30a5p-MO, 5�-AGCTTCCAGT -CGAGGATGTTTACAG-3�; miR-34b-MO, 5�-ACAATCAGCTAACT -ACACTGCCTGA-3�; and Std-MO, 5�-CCTCTTACCTCAGT TAC -AATTTATA-3�. Antisense MOs were diluted to 1 mM. The Dgcr8-MOrescue construct pCS2-Dgcr8* was generated by PCR mutagenesis,introducing six nucleotide changes so that the construct was no longertargeted by Dgcr8-MO, but was translated into a protein of identical aminoacid sequence. pCS2-Lhx1* was sub-cloned from pXH32 (a kind gift fromDr I. Dawid, NIH, USA), changing six nucleotides in the recognition site ofLhx1-MO and removing the entire 3�UTR. The pCS2-Dicer-GFP and pCS2-Dgcr8-GFP constructs were generated by PCR from pCS2-xDicer andpCS2-xDgcr8 and subcloned into pCS2-GFP. For synthetic mRNA, allplasmids were linearized with NotI and transcribed with SP6 RNApolymerase using the mMessage mMachine (Ambion).

For all injections, a total of 8 nl of MO solution was injected radially atthe 2- to 4-cell stage into Xenopus embryos. For the rescue experiments,these injections were followed by a single injection of synthetic mRNA intothe marginal zone. In the miR-30 reporter assays, a subset of embryos wasinjected with 160 fmol miRNA duplex (miR-30a-5p or miR-17) into theanimal region at the 2-cell stage in the presence or absence of 3.2 pmol miR-30a5p-MO or miR-34b-MO. These embryos and uninjected embryos werethen injected at the 4-cell stage with a total of 8 ng synthetic mRNA(pXEXGal-Lhx1-3�UTR or pXEXGal-miR-30a-5p) animally into twoblastomeres. Embryos were processed at gastrula stage for lacZ stainingusing standard procedures.

In situ hybridizationIn situ hybridizations were carried out as described previously (Belo et al.,1997). To generate antisense probes, plasmids were linearized andtranscribed as follows: pGEM-T-Cadherin16, ApaI/SP6 (GenBankaccession number GQ499200); pBC-ClC-K, EcoRI/T7 (Vize, 2003);pGEM-T-HNF1, Asp718/T7 (Vignali et al., 2000); pSP64TS-Lim1,XhoI/T7 (Carroll et al., 1999); pSK-1-Na/K-ATPase, EcoRI/T7 (Tran et al.,2007); pCMV-SPORT6-xNbc-1, SalI/T7 (Zhou and Vize, 2004); pSK-NCC,EcoRI/T7 (Tran et al., 2007); pSK-NKCC2, SmaI/T7 (Tran et al., 2007);

pSK-Pax-2, XbaI/T7 (Carroll et al., 1999); pSK-Pax-8, BamHI/T7 (Carrollet al., 1999); pCMV-SPORT6-ROMK, EcoRI/T7 (Tran et al., 2007); pCMV-SPORT6-xSGLT1-K, SalI/T7 (Zhou and Vize, 2004); pGEM-T-Slc7a13,SalI/T7 (nt 862 to 1480 of GenBank accession number BC060020). Thefollowing LNA-modified degenerate and specific oligomers weresynthesized by Integrated DNA Technologies and labeled with digoxigeninas previously described (Wienholds et al., 2005): miR-138, 5�-CGLNA -GCCLNA TGALNATTCLNAACALNAACALNACCALNAGCT-3�; miR-200family, 5�-CRKCLNADTTLNAACCLNAMGRCLNAAGTLNARTTLNAA-3�;miR-30 family, 5�-SALNAGTLNASDRGLNAGATLNAGTTLNATACLNAA-3�;miR-30a-5p, 5�-CTTLNACCALNAGTCLNAGAGLNAGATLNAGTTLNA -TACLNAA-3�; miR-30b, 5�-AGCLNATGALNAGTGLNATAGLNAGATLNA -GTTLNA T ACLNA A-3�; miR-30c, 5�-GCTGLNAAGALNAGTGLNATAGLNA -GATLNAG TTLNA TACLNAA-3�; miR-489, 5�-GCLNATGCLNAC ATLNA - ATALNAT GTLNA GGTLNAGTCLNAATT-3�.

Histology, immunohistochemistry and TUNEL stainingFor histological staining, Xenopus embryos were fixed in Bouin’sFixative, dehydrated, embedded in paraplast, sectioned at 7 m, dewaxed,and stained with Hematoxylin and Eosin. For immunohistochemistry,embryos were fixed in Dent’s fixative (4:1 methanol:DMSO). For whole-mount immunostaining, embryos were incubated overnight with 3G8 and4A6 monoclonal antibodies (Vize et al., 1995) followed by incubationwith a horseradish peroxidase-coupled anti-mouse IgG and developedusing the ImmPACT DAB Kit (Vector Laboratories). Forimmunohistochemistry on sections (i.e. the proliferation analysis), theembryos were embedded in paraplast, sectioned at 25 m and probed withanti-phospho-Histone H3 (Upstate Biotechnology). For TUNEL staining,Xenopus embryos were fixed in MEMFA, dehydrated, embedded inparaplast, sectioned at 10 m and analyzed using the DeadEndColorimetric TUNEL System (Promega).

microRNA profilingmiRNA profiling was performed using the miRCURY LNA miRNA Arrays(Exiqon) consisting of all miRNA in all organisms as annotated in miRBaserelease 8.1. The results have been deposited to Gene Expression Omnibus(accession number GSE18609). For the candidate miRNA approach, totalRNA was isolated using RNA STAT-60 (Tel-Test). RT-PCR was performedusing a protocol described previously for miRNAs (Chen, C. et al., 2005)with minor modifications. The looped RT primer was modified to contain arandom hexamer at the 3� end (5�-GTCGTATCCAGTGCAGGG TCCGAG -GTATTCGCACTGGATACGACNNNNNN-3�). A common reverse primer(5�-GTGCAGGGTCCGAGGT-3�) and miRNA-specific forward primers(see Table S1 in the supplementary material) were used for PCRamplification and the products were analyzed on agarose gels.

miRNA reporter constructs and miRNA duplexesThe pXEXGal-miR-30a-5p construct, containing a triplet of miR-30a-5precognition sites (underlined in the oligomers), was designed as described(Giraldez et al., 2006) using the following four oligomers: miR-30a-5p-X,5�-TCGAGAATCTAGATTCCAGTCGAGGATGTTTACATAGTA-3�;miR-30a-5p-Y, 5�-TTCCAGTCGAGGATGTTTACATAGTATTCCAG -TCGAGGATGTTTACACTGCA-3�; miR-30a-5p-W, 5�-TCCTCGACT -GGAATACTATGTAAACATCCTCGACTGGAATCTAGATTC-3�; andmiR-30a-5p-Z, 5�-GTGTAAACATCCTC GACT GGAATACTATGTAA -ACA-3�. Oligomers were annealed and cloned first into pAcGFP (ClontechLaboratories) and then subcloned into pXEXGal (a kind gift from R.Harland, University of California, Berkeley, CA, USA) using XbaI andAsp718. The pXEXGal-Lhx1-3�UTR and pmir-GLO-Lhx1-3�UTRconstructs were prepared by amplifying a part of the 3�UTR of Xlim1 (5�-GGGCTAGCTGTTAGGTGGTGCACAGGAC-3�, 5�-GAGGTACC CC -TGTTTGGGTCTATGTAAATC-3�) and subcloning it into pXEXGal usingXbaI and Asp718 or into pmir-GLO (Promega) using NheI and SalI. pmir-GLO-Lhx1-3�UTR-mut was generated using the QuikChange Site-DirectedMutagenesis Kit (Stratagene), changing the core of the two miR-30 bindingsites from 5�-TGTTTAC-3� to 5�-TCTATTC-3�.

For the synthetic miRNA duplexes, two RNA/DNA hybrid oligomerswith the following sequences were synthesized (Integrated DNATechnologies) and annealed: miR-30a-5p duplex, 5�-rUrCrCrArGrUr -

RESEARCH ARTICLE Development 136 (23)

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CrGrArGrGrArUrGrUrUrUrArGrAAG-3� and 5�-rUrGrUrArArArCrAr -UrCrCrUrCr Gr ArCrUrGrGrAAG-3�; miR-17 duplex, 5�-rArCrCrUr -GrCrArCrUrGrUrArArGrCrArCrUrUrUrGTT-3� and 5�-rCrArArArGrUr -Gr CrUrUrArCrArGrUrGrCrArGrGrUAG-3�.

For the miRNA expression constructs, pri-miRNA precursors including~200 bp flanking sequences (to facilitate proper folding and cleavage to themature miRNAs) were amplified by PCR and subcloned into pCS2 togenerate pCS2-miR-30a-5p (5�-GCTTTGCAGTTTACAGAATGT-3� and5�-CCTCGAGTCTGTGGTTTTGAAGTTGTTT-3�) and pCS2-miR-17 (5�-GAACTTCTGGCTATTGGCTCCTC-3� and 5�-CCTCGAGCACGCAG -CACCAGCAG-3�). For the luciferase assays, HEK 293T cells weretransfected using Lipofectamine 2000 (Invitrogen) with 50 ng of the pmir-GLO constructs and 1 g of the miRNA vectors and processed using theDual-Luciferase Reporter Assay (Promega) after 48 hours.

RESULTSGlobal inhibition of miRNA biogenesismiRNAs undergo multiple steps of post-transcriptional processingand are non-functional until the mature miRNA is loaded into theRISC complex (Kloosterman and Plasterk, 2006; Bushati andCohen, 2007; Stefani and Slack, 2008). As a first step to explore theoverall role of miRNAs during pronephric kidney development, wedepleted two key proteins in this process, Dicer and Dgcr8, usingantisense morpholino oligomers (MOs). Xenopus embryos wereinjected at the 2- to 4-cell stage in all four blastomeres with differentamounts of Dicer-MO or Dgcr8-MO to determine the sub-optimaldoses that allowed survival until tailbud stage. Interestingly, the twoantisense MOs did not behave identically. Injection of a total of 6.4pmol Dicer-MO permitted reasonable survival of the embryos,whereas only a 6-fold lower amount of Dgcr8-MO (1.07 pmol) wastolerated. As shown in Fig. 1A-A�, the Dicer-MO- and Dgcr8-MO-injected embryos at stage 43 were generally malformed, displayedeye defects and developed edema, a condition often caused by animpaired osmoregulatory function of the pronephros (Howland,1916). Histological sections at stage 42 showed an enlarged bodycavity, malformations of the endoderm, a reduced neural tube,disintegrating somites and a reduction of the pronephric tubules(Fig. 1B-B� and data not shown). Although these morphants had awide range of defects, we focused in this study on the developmentof the pronephric kidney.

To address whether specific pronephric malformations could bedetected, embryos were examined at stage 40 before the onset ofgross morphological abnormalities and the formation of edema. Thepronephros was visualized by immunostaining with two antibodies,3G8 and 4A6, that specifically stain the proximal tubules or thedistal tubules and the pronephric duct, respectively (Vize et al.,1995). In agreement with the histology, both Dicer-MO- and Dgcr8-MO-injected embryos displayed a reduced area of 3G8-positiveproximal tubules (Fig. 1C-C�). Interestingly, 4A6 staining wasdetected in the distal tubules, but was absent in the pronephric duct(Fig. 1D-D�). This was not due to a general disappearance of thepronephric duct, as whole-mount in situ hybridization showedexpression of the pan-pronephros marker 1-Na/K-ATPasethroughout the tubules and duct (Fig. 1E-E�). The absence of 4A6staining was probably a result of delayed terminal differentiation ofthe renal epithelial cells in the pronephric duct, as Dgcr8-MO-injected embryos gradually recovered 4A6 staining at later stages(see Fig. S2 in the supplementary material).

The highly similar pronephric defect caused by targeting of twoindependent proteins involved in miRNA biogenesis (Dicer andDgcr8) suggested that this phenotype was due to the lack ofmiRNAs. It was not observed in embryos injected with a standardcontrol morpholino (Std-MO, Fig. 1F and data not shown).

3929RESEARCH ARTICLEmiR-30 and kidney development

Fig. 1. Inhibition of miRNA biogenesis results in pronephricabnormalities. (A-E�) Xenopus embryos were injected with 6.4pmol Dicer-MO or 1.07 pmol Dgcr8-MO and compared withuninjected sibling embryos by morphology at stage 43 (A-A�),histology with Hematoxylin and Eosin at stage 42 (B-B�),immunostaining with 3G8 and 4A6 at stage 40 (C-D�), and bywhole-mount in situ hybridization for 1-Na/K-ATPase at stage 39(E-E�). Arrowheads indicate edema formation (A�,A�) and the loss of4A6 staining in duct (D�,D�). en, endoderm; no, notochord; nt,neural tube; pn, pronephros; s, somites. (F)Quantification of 4A6staining in the pronephric duct, comparing uninjected controlembryos with embryos injected with a standard control MO (Std-MO), Dicer-MO, Dgcr8-MO, a combination of Dicer-MO and Dgcr8-MO, or Dgcr8-MO together with Dgcr8* mRNA at stage 40. Thegraph represents the summary of at least three independentexperiments. The number (N) of embryos analyzed is indicatedabove the bars. Black, normal expression; gray, partial expression;white, no expression. (G)RT-PCR analysis of multiple miRNAs afterDicer and Dgcr8 knockdown at stage 35. SNO-412 served asloading control. D

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Moreover, the efficacy of the Dicer-MO and the Dgcr8-MO wasdemonstrated using GFP-fusion protein constructs that contained theMO binding sites (see Fig. S1 in the supplementary material).Whereas Xenopus embryos injected animally with 8 ng mRNAencoding Dicer-GFP or Dgcr8-GFP displayed GFP expression atlate gastrula stage, this fluorescence was lost upon co-injection withDicer-MO or Dgcr8-MO, respectively.

To further underscore the specificity of the knockdowns, a rescueexperiment was performed using a Dgcr8* mRNA that has sixnucleotide mismatches in the recognition site of the Dgcr8-MO andis resistant to its activity. Injection of synthetic Dgcr8* mRNA intoall four blastomeres of a 4-cell stage embryo resulted in severedevelopmental defects and did not allow us to perform rescueexperiments (data not shown). Thus, we decided to use a strategysimilar to the one previously described by Tran et al. (Tran et al.,2007). Xenopus embryos were injected at the 2-cell stage radiallywith Dgcr8-MO. Subsequently, a subset of these embryos wasinjected with Dgcr8* mRNA into a single blastomere at the 4-cellstage. Embryos were grown until stage 40 and processed for whole-mount immunostaining with 4A6. As described above, 4A6 stainingwas lost in the pronephric duct upon injection of Dgcr8-MO, but wasnow recovered upon co-injection of Dgcr8* mRNA (Fig. 1F).Unfortunately, we could not perform a similar rescue experiment forthe Dicer-MO as we were not successful in isolating a functionalallele of Xenopus Dicer.

Another argument for the specificity of the phenotype was thecooperativity of Dicer and Dgcr8. Whereas sub-optimal doses of thetwo antisense MOs (3.2 pmol Dicer-MO or 0.5 pmol Dgcr8-MO) didnot affect 4A6 staining, the combination of both resulted in a more-than-additive reduction in 4A6 staining in the pronephric duct (Fig.1F). Finally, semi-quantitative RT-PCR at stage 35 showed that thelevels of multiple mature miRNAs were reduced, but – as expectedfrom the suboptimal doses of the MOs – not completely eliminated(Fig. 1G). Based on the identical results of the two MOs, we decidedto primarily use the Dgcr8-MO for the remainder of the study.

The pronephros is organized along its proximal-distal axis in amanner that is highly similar to the metanephric nephron (Zhouand Vize, 2004; Tran et al., 2007; Raciti et al., 2008). To addresswhether inhibition of miRNA biogenesis would affect thisorganization, Dgcr8-MO-injected and uninjected control embryoswere cultured until stage 39 and processed for whole-mount insitu hybridization. A panel of segment-specific terminaldifferentiation genes (Fig. 2H) showed that in the Dgcr8knockdown, all pronephric segments were present, but theexpression domains of individual markers were affected (Fig. 2I).The domains of the proximal tubule markers SGLT1-K andSlc7a13 were reduced (Fig. 2A,A� and data not shown). NKCC2,a marker for the intermediate tubules and part of the distal tubule,was less convoluted and was extended posteriorly (Fig. 2B,B�).The expression of NBC1 in the distal tubule was shorter and moreabutted to the proximal tubule (Fig. 2C,C�). ClC-K, Cadherin-16and ROMK, three genes expressed in the intermediate and distaltubule as well as in the pronephric duct, showed a dramaticreduction in the anterior part of their expression domains (Fig.2D-F�). Only NCC, a marker for the distal tubule and thepronephric duct, appeared relatively unchanged (Fig. 2G,G�).Similar results were observed in embryos injected with the Dicer-MO (see Fig. S3 in the supplementary material).

Together, these data suggested that miRNAs play an importantrole during pronephros development. Interestingly, they do not seemto regulate the early inductive events, but rather later phases, suchas the outgrowth and morphogenesis of the individual tubulesegments and the timing of pronephric duct differentiation.

Screening for miRNAs expressed in the kidneyNext, we set out to identify those miRNAs that could exert thisregulatory function in pronephros development, pursuing twoparallel approaches. First, mouse metanephric kidneys fromembryonic day (E) 14.5 and postnatal day (P) 1 were screenedusing the miRCURY LNA Array miRNA profiling service from


Fig. 2. Inhibition of miRNA biogenesisaffects patterning of the pronephros. (A-G�) Whole-mount in situ hybridization formarkers of terminal pronephros differentiationon uninjected and Dgcr8-MO-injected Xenopusembryos at stage 39. (A,A�) SGLT1-K; (B,B�)NKCC2; (C,C�) NBC1; (D,D�) ClC-K; (E,E�)Cadherin-16; (F,F�) ROMK; (G,G�) NCC.Arrowheads indicate a reduction in theintermediate and distal tubular domain.(H,I)Schematics illustrating the differentpronephric regions and their correspondingmarker gene expression (H) and the phenotypeof Dicer-MO- or Dgcr8-MO-injected embryosshowing a reduction in the proximal andintermediate tubules as well as in distal tubuleDT1 (I).

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Exiqon to identify not only miRNAs present in the kidney, butalso those that undergo differential regulation during development(see Fig. S4A in the supplementary material). Second, candidatesfor kidney-enriched miRNAs were selected from previouspublications on human, mouse, rat and zebrafish (Sun et al., 2004;Naraba and Iwai, 2005; Wienholds et al., 2005) and re-analyzedby RT-PCR, comparing four organs of adult mice and differentstages of Xenopus development (see Fig. S4B in thesupplementary material). From these two data sets, severalmiRNAs/miRNA families were selected for subsequentexpression profiling. In situ hybridization on mouse kidneysections at E14.5 and P0 and Xenopus embryos at stages 34, 39and 41 were performed using locked nucleic acid (LNA)-modified probes (Fig. 3 and data not shown). To accelerate thescreen, miRNA families were initially studied using degenerateLNA oligomers. Individual family members were only analyzedonce expression of the family was confirmed in the kidney.

Although many of the candidates were expressed in the kidney,the miR-30 family (miR-30a-5p, miR-30b, miR-30c-1, miR-30c-2,miR-30d and miR-30e) was the most striking. In particular, itsexpression in Xenopus was rather specific for the pronephros incomparison with other miRNAs, such as the miR-200 family, thatwere strongly expressed in various tissues besides the pronephros

(Fig. 3B). Therefore, individual members of the miR-30 family wereanalyzed using three LNA-modified probes: miR-30a-5p (whichcross-reacts with miR-30d and miR-30e), miR-30b and miR-30c(which recognizes miR-30c-1 and miR-30c-2). All family membersshowed a very similar expression pattern, with miR-30a-5pappearing slightly earlier than the other two (Fig. 3C and data notshown). They were strongly expressed in the pronephros and weaklyin neural tube, somites and head structures (Fig. 3B,C and data notshown). No staining was detected before stage 30 (data not shown)and pronephric expression was first detected around stage 34.

Knockdown of miR-30a-5p activityNext, we sought to determine whether the miR-30 familycontributed to the kidney phenotype observed in embryos injectedwith Dicer-MO or Dgcr8-MO. We targeted the miR-30 family byantisense MOs similar to those used in recent reports in zebrafish(Flynt et al., 2007; Eberhart et al., 2008). Since miR-30a-5p wasthe earliest expressed family member (Fig. 3B,C), we designed amiR-30a-5p-specific antisense MO (miR-30a5p-MO). This MOmost likely also targeted miR-30d and miR-30e as they differ byonly one nucleotide from miR-30a-5p. A second antisense MOwas designed in an identical fashion, but directed against anunrelated miRNA, miR-34b (miR-34b-MO), to serve as an internal


Fig. 3. Kidney expression of miRNAs. (A)In situ hybridizations of mouse kidney sections at E14.5 and P0 using LNA-modified oligonucleotideprobes. miRNA families were studied using degenerate probes that recognize all family members. (B)Whole-mount in situ hybridization of Xenopusembryos at stages 34, 39 and 41. Arrows indicate pronephros. Insets show magnified view of pronephros. (C)Transverse section of Xenopusembryos after whole-mount in situ hybridization with probes recognizing the entire miR-30 family or individual members. Insets show enlargementsof the pronephros.

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control. To test the efficacy of miR-30a5p-MO, the followingstrategy was used. Xenopus embryos were injected in the animalpole at the 2-cell stage with 160 fmol miR-30a-5p duplex in thepresence or absence of either 3.2 pmol miR-30a5p-MO or miR-34b-MO. At the 4-cell stage, these embryos, as well as uninjectedcontrol embryos, were injected with 4 ng of a -galactosidasereporter containing three copies of an optimal miR-30a-5pbinding site in its 3�UTR. At gastrula stage, embryos wereprocessed for lacZ staining. As shown in Fig. 4A,A�, the miR-30a-5p duplex downregulated the activity of the -galactosidasereporter. Co-injection of miR-30a5p-MO reversed this effect andlacZ expression was regained. This was specific, as it was notobserved when miR-34b-MO was injected.

Next, to analyze the role of miR-30a-5p during pronephrosdevelopment, Xenopus embryos were injected with 3.2 pmol miR-30a5p-MO at the 2- to 4-cell stage and cultured until late tailbudstage. Interestingly, these embryos exhibited pronephric defectsvery similar to those observed in Dicer-MO- or Dgcr8-MO-injected embryos. miR-30a5p morphants developed severe edemaafter stage 43 and exhibited reduced pronephric tubules whenexamined by histology (Fig. 4B-C�). Moreover,immunohistochemistry with 4A6 and 3G8, as well as whole-mount in situ hybridization with the same panel of marker genesas in Fig. 2, revealed reduced proximal tubules, delayeddifferentiation of the pronephric duct and defects in theorganization of the individual tubular segments (Fig. 4D-H� anddata not shown). The similarity of the two phenotypes (Dicer-MOor Dgcr8-MO and miR-30a5p-MO) was striking, but clearlyspecific. Microinjections targeting another kidney miRNA (miR-34-MO) did not result in any of these pronephric defects (data not

shown). Unfortunately, a rescue experiment turned out to beunfeasible because the synthetic miR-30a-5p duplex wasinsufficiently stable to persist until late stages of development(data not shown).

These data demonstrated that the miR-30 family is important forpronephros development and that its inhibition phenocopies most ofthe pronephric defects observed upon global inhibition of miRNAbiogenesis.

miR-30 regulates proliferationOne important function of miRNAs is to regulate the balancebetween proliferation and apoptosis (Chivukula and Mendell,2008). Such an imbalance could therefore be responsible forcertain aspects of the miR-30a5p-MO phenotype (e.g. the reducedpronephric tubules). However, TUNEL staining did not detect anincrease in apoptosis in the pronephros at stage 40 (Fig. 5A,A�).By contrast, mitotic cells positive for phospho-Histone H3 weredramatically decreased (Fig. 5B,B�,C). Inhibition of proliferationwas observed only at stage 40, and not at stage 37 (Fig. 5C) whenchanges in gene expression were already detectable (data notshown). To address the contribution of decreased proliferation tothe phenotype, uninjected Xenopus embryos were treated withaphidicolin and hydroxyurea from stage 33/34 until stage 40 toabolish all cell divisions (Harris and Hartenstein, 1991). Eventhough these embryos were devoid of mitotic cells (Fig. 5B�),they did not exhibit any differences in the pattern of 4A6 stainingor in Cadherin-16 and NKCC2 expression (Fig. 5D-F�). Thissuggested that although proliferation is clearly affected byknockdown of miR-30 activity, it was not responsible for thephenotypic changes seen with miR-30a5p-MO.


Fig. 4. miR-30a-5p knockdown and globalinhibition of miRNA biogenesis have very similarkidney phenotypes. (A)To determine the specificityof miR-30a5p-MO, three consecutive miR-30a-5pbinding sites (BS) were introduced in the 3�UTR of alacZ reporter. Xenopus embryos were injected withthe reporter in the presence or absence of miR-30a-5p duplex and miR-30a5p-MO and processed for lacZstaining at gastrula stage. Injections with miR-34b-MO served as a specificity control. (A�)Quantificationof the lacZ staining in embryos: white, no staining;gray, partial staining; black, strong staining. Thenumber of embryos analyzed in three differentexperiments is indicated above the bars.(B-H�) Xenopus embryos injected with miR-30a5p-MOand uninjected controls were analyzed by morphology(B,B�), histology (C,C�), immunohistochemistry with3G8 and 4A6 (D-E�), and whole-mount in situhybridization for Cadherin-16 (Cad-16; F,F�), ClC-K(G,G�) and NKCC2 (H,H�). Arrowheads indicate thepresence of edema (B�), loss of 4A6 staining in thepronephric duct (E�), and the shortening of the tubulesegments IT1, IT2 and DT1 (F�,G�,H�). en, endoderm;no, notochord; nt, neural tube; pn, pronephros;s, somites.

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miR-30 regulates Xlim1/Lhx1 activityAll the genes analyzed so far are markers for terminaldifferentiation in the pronephros. Next, we tested whether miR-30a5p-MO affected upstream transcription factors. Xenopusembryos injected with miR-30a5p-MO and uninjected controlswere processed for in situ hybridization with Pax2, Pax8, Hnf1-and Xlim1 at stage 39. As shown in Fig. 6A-C�, the expression

levels of Pax2, Pax8 and Hnf1- mRNA were unchanged, eventhough their domains reflected the altered pronephrosarchitecture, as seen with terminal differentiation markers such as1-Na/K-ATPase (Fig. 1E-E�). By contrast, Xlim1 mRNA waselevated in the pronephric duct (Fig. 6D,D�). Loss of miRNAactivity not only results in increased translation, but can alsoincrease mRNA stability (Giraldez et al., 2006), suggesting that


Fig. 5. Apoptosis and proliferation do not contribute to the kidney phenotype. (A,A�) TUNEL staining of transverse sections from uninjectedand miR-30a5p-MO-injected Xenopus embryos at stage 39. TUNEL-positive cells appear brown and are indicated by arrows. Note that nosignificant differences were observed by analyzing many serial sections of the pronephros. Several embryos and multiple independent experimentswere analyzed. (B-B�) Immunofluorescence analysis of proliferation with an anti-phospho-Histone H3 antibody comparing transverse sections ofuninjected control embryos, embryos injected with miR-30a5p-MO and embryos treated with aphidicolin and hydroxyurea (APC+HU) at stage 40.(C)Quantification of phospho-Histone H3-positive cells in the pronephros of uninjected (dark gray) and miR-30a5p-MO-injected (light gray) embryosat stages 37 and 40. Consecutive sections covering the entire pronephric tubular area were counted. Numbers are the average of at least fourdifferent embryos from two independent experiments. Error bars represent s.d. (D-F�) Immunostaining and in situ hybridization of untreated andAPC+HU-treated embryos with 4A6 (D,D�), Cadherin-16 (Cad-16; E,E�) and NKCC2 (F,F�). Note the absence of any patterning differences, eventhough proliferation was completely inhibited.

Fig. 6. Xlim1 is regulated by miR-30a-5p.(A-D�) Whole-mount in situ hybridization for Pax2(A,A�), Pax8 (B,B�), Hnf1- (C,C�) and Xlim1 (D,D�),comparing uninjected and miR-30a5p-MO-injectedXenopus embryos at stage 39. (E-G�) Whole-mountin situ hybridization for Xlim1 mRNA of uninjectedand miR-30a5p-MO-injected Xenopus embryos atstages 31, 35 and 39. Note that Xlim1 expression ismaintained at a higher level in miR-30a5p-MO-injected embryos after stage 35, when endogenousexpression of miR-30a-5p can first be detected(arrows in D�,G�). (H,H�) Immunohistochemistrywith 4A6 of stage 40 embryos injected with 4 pgpCS2-Lhx1* DNA into one blastomere at the 4-cellstage, comparing the left with the right side.Arrows indicate changes in 4A6 staining. Ectopicexpression of Lhx1 mRNA transcribed from thepCS2-Lhx1* was confirmed by in situ hybridization(data not shown).

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Xlim1 could be a direct target of miR-30 activity. In Xenopus,Xlim1 is expressed in the pronephric anlage as early as stage 13(Taira et al., 1994; Carroll and Vize, 1999), whereas the miR-30family is first detected at stage 34 (Fig. 3B,C). If miR-30 directlydownregulates Xlim1, the loss of miR-30 should stabilize Xlim1mRNA levels as soon as miR-30 normally becomes expressed inthe pronephros. Indeed, comparison of miR-30a5p-MO-injectedembryos with uninjected sibling controls at stages 31, 35 and 39showed that the expression of Xlim1 was nearly indistinguishableat stage 31, but was maintained at higher levels in embryos withreduced miR-30 activity as early as stage 35 (Fig. 6E-G�).

Increased Xlim1 expression could be part of the miR-30a5p-MOphenotype. To test this, we elevated Xlim1 levels without changingmiR-30 activity by a one-sided injection with 4 pg pCS2-Lhx1*DNA. This construct contains an optimized Kozak sequence andlacks the entire 3�UTR. Xenopus embryos were injected at the 2-cellstage, cultured until stage 40 and processed for 4A6immunostaining. Fifty-five percent of the embryos (n98) displayedvarious degrees of change in 4A6 staining upon comparison of theuninjected and DNA-injected sides (Fig. 6H,H� and see Fig. S5 inthe supplementary material). This result agreed with our hypothesisthat increased levels of Xlim1 interfere with terminal differentiationof the pronephros.

To test whether Lhx1 mRNA is a direct target of the miR-30family, we first performed an in silico analysis to detect putativemiRNA binding sites. Unfortunately, the 3�UTR of Lhx1 had notbeen described. So we initially used a prediction algorithm based ongenomic sequences (DIANA microT version 3.0). This approachidentified potential miRNA binding sites in mouse Lhx1 includingtwo strong miR-30 binding sites (Fig. 7A). Moreover, these siteswere present in authentic Lhx1 transcripts, as they were found inESTs of human, mouse and Xenopus, as well as in the previouslyuncharacterized 3�UTR of a full-length Xenopus Xlim1 cDNA clone(Taira et al., 1992) (GenBank accession number GQ485551). Asshown in Fig. 7B, the two miR-30 binding sites were completelyconserved.

To address the functionality of these two sites, the 3�UTR ofXenopus Xlim1 was subcloned downstream of the lacZ reporter.Synthetic mRNA was injected into the animal region of Xenopusembryos in the absence or presence of a synthetic miR-30a-5p ormiR-17 duplex and processed for lacZ staining at gastrula stage.Injection of the reporter construct alone showed strong lacZ staining,but was significantly reduced upon co-injection of the miR-30a-5pduplex (Fig. 7C-F). This effect was specific, as injection of a miR-17 duplex that does not have a predicted binding site within the3�UTR did not alter the lacZ staining.

Next, in order to quantify the miR-30a-5p effects, HEK 293Tcells were transfected with a dual luciferase reporter constructharboring the 3�UTR of Xlim1 (pmir-GLO-xLhx1-3�UTR) in thepresence or absence of expression constructs expressing miR-30a-5p or miR-17 under the control of the CMV promoter (pCS2-miR-30a-5p, pCS2-miR-17). As shown in Fig. 7G, miR-30a-5preduced luciferase expression by 35%. miR-17 did not have anyeffect. Moreover, when both miR-30 binding sites in the 3�UTRof Xlim1 were mutated (pmir-GLO-xLhx1-3�UTR-mut), therepression by miR-30a-5p was lost. Thus, repression of Xlim1 bymiR-30 was mediated by the two miR-30 binding sites identifiedin its 3�UTR.

Together, these data make a compelling argument that geneexpression in the kidney must be tightly controlled to allow properpronephric kidney development and that the miR-30 miRNA familyplays an important role in this regulation.

DISCUSSIONmiRNAs are involved in a variety of biological processes and thenumber of isolated or predicted miRNAs continues to increase.However, the in vivo significance of only a few miRNAs has been


Fig. 7. Xlim1/Lhx1 is a direct target of miR-30a-5p-mediatedrepression. (A)In silico analysis of putative miRNA binding sites in the3�UTR of mouse Lhx1 using DIANA microT (version 3.0 using strictthreshold conditions). Blue circles represent predicted miRNA bindingsites and the red boxes indicate two strong miR-30 binding sites in theLhx1 3�UTR. (B)Sequence alignment showing the conservation of themiR-30 binding sites in human, mouse and Xenopus Xlim1/Lhx1. ThemiR-30 seed sequences are indicated in red. (C-F)-galactosidasereporter assay of Xenopus embryos injected with pXEXGal-Lhx1-3�UTRmRNA in the presence or absence of a miR-30a-5p or a miR-17 duplexat gastrula stage. (G)Luciferase reporter assay of HEK 293T cellstransfected with pmir-GLO-xLhx1-3�UTR and pmir-GLO-xLhx1-3�UTR-mut in the presence of pCS2, pCS2-miR-30a-5p or pCS2-miR-17. Valueswere corrected for the expression of Renilla luciferase and calculated asfold change compared with the pCS2 control. Multiple independentexperiments were averaged and s.d. is indicated. (H,H�) Model for theregulation by miR-30 of Lhx1 expression. During kidney development,Lhx1 mRNA and protein levels gradually decline, whereas miR-30a-5plevels increase (H). Upon injection of the miR-30a5p-MO, Lhx1 levelspersist, impairing terminal differentiation (H�).

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elucidated so far (Stefani and Slack, 2008). We now reveal the roleof the miR-30 family during pronephric kidney development inXenopus. Knockdown of miR-30a-5p by antisense MOs resulted ina complex phenotype characterized by a delay in differentiation,shortening of individual nephron segments and reducedproliferation. Intriguingly, these changes were very similar to thekidney phenotype observed when global miRNA maturation wasinhibited using Dicer-MO or Dgcr8-MO. This argues that the miR-30 family has a central role in nephrogenesis. Such an interpretationfinds support in three recent studies of miRNAs in podocytes thatshow that Dicer is required for miR-30 expression (Harvey et al.,2008; Ho et al., 2008; Shi et al., 2008). Nevertheless, the central roleof miR-30 is also puzzling. Our expression profiling identifiedseveral other miRNAs present in the kidney of frog and mouse,including the miR-200 family, miR-489 and miR-138. It is likely thatthey too will have important functions in the kidney, but they mightpromote different aspects of kidney development that were notaddressed with the panel of markers used in this study. Indeed, aparallel study in our laboratory has identified the miR-17 family asa crucial regulator of polycystic kidney disease genes in Xenopusand mouse (our unpublished results).

A second interesting aspect of this study was that global inhibitionof miRNA biogenesis did not interfere with the induction of thepronephros. The initial expression of transcription factors patterningthe intermediate mesoderm (such as Pax2, Pax8, Xlim1 and Hnf1-) was unaffected (Fig. 6 and data not shown). Defects only becameapparent when these transcription factors are downregulated toallow terminal differentiation of the pronephros (Carroll et al.,1999). At this junction, miRNAs might provide some plasticity.Indeed, several miRNAs, such as the miR-200 family, are crucialduring the epithelial-mesenchymal transition, when a differentiatedepithelial cell is converted into a mesenchymal cell type (Cano andNieto, 2008). Interestingly, miR-200 was also identified during ourexpression profiling as a kidney-enriched miRNA and it will beinteresting to address their function on E-Box genes, such as Zeb1and Zeb2, during kidney development.

One of the most challenging aspects in studying miRNAs is theprediction and verification of genes regulated by a given miRNA(Bartel, 2009). In this study, we identified the LIM-class homeoboxgene Xlim1/Lhx1 as a target of miR-30 activity. This argument isbased on four observations. (1) The 3�UTR of human, mouse andXenopus Lhx1 contains two highly conserved miR-30 binding sites.(2) miRNA reporter assays showed that this 3�UTR was repressedby miR-30a-5p and this depended on the presence of these twobinding sites. (3) Downregulation of Xlim1 mRNA in the Xenopuspronephros coincided with the appearance of miR-30 expression. (4)Overexpression of Xlim1 mimicked the loss of 4A6 stainingobserved in the miR-30 knockdown. Based on these data, wehypothesize that Xlim1 mRNA is an important target of miR-30activity and sharpens the time window of Xlim1 expression in thekidney (Fig. 7H,H�). In the presence of miR-30a-5p, the expressionof Xlim1 starts to decline at about stage 35 (Fig. 7H). However, inthe absence of miR-30a-5p activity, this window of Xlim1expression is extended, contributing to the delayed terminaldifferentiation of the renal epithelial cells (Fig. 7H�).

The observation that Xlim1/Lhx1 is under miRNA control is evenmore important because it is a central player in kidney developmentand exhibits a dynamic expression pattern that needs to be tightlycontrolled (Dressler, 2006). In Xenopus, Xlim1 mRNA is initiallyfound throughout the pronephric tubules and duct. As tubules beginto lumenize, expression becomes restricted to the nephrostomes andthe late distal tubule (Carroll et al., 1999; Tran et al., 2007).

Similarly, in mouse, Lhx1 is expressed in the nephric duct, the tipsof the ureteric buds, the pretubular aggregates, the comma- and S-shaped bodies and even the podocytes of the immature glomerulus(Barnes et al., 1994; Fujii et al., 1994; Kobayashi et al., 2005).Moreover, all these individual domains are important for properkidney development and tubular morphogenesis (Kobayashi et al.,2005; Pedersen et al., 2005). An miRNA-based regulation providesthe possibility to fine-tune Xlim1/Lhx1 expression independently oftranscriptional control. Based on the data presented here, the miR-30 family constitutes such a regulator. It controls terminaldifferentiation of the Xenopus pronephros by modulating Xlim1.Because miR-30 expression appears at around stage 34, it cannotcontrol all phases of pronephric expression of Xlim1. The earlyregulation might be accomplished by other miRNAs. Our analysisof the Lhx1 3�UTR identified two additional miRNA binding sitesfor miR-96/182 (Fig. 7A), an miRNA family that, based onmicroarray data (data not shown), is expressed during early mousekidney development.

Obviously, Xlim1/Lhx1 will not be the only target of miR-30activity. In silico analyses suggest many other potential targets. Theosmoregulatory transcription factor NFAT5 (TonEBP) (Ho, 2006)has three conserved miR-30 binding sites. Similarly, the mRNAs ofa large number of solute carrier (SLC) proteins (He et al., 2009) havemiR-30 binding sites in their 3�UTR. These proteins are involved inrenal homeostasis. In the future, it will be interesting to see whetherloss of miR-30 activity in the adult has consequences for kidneyfunction. This might bring forth examples of dual-functionmiRNAs: although they direct patterning during early development,they are later required to maintain organ function.

AcknowledgementsWe thank Drs S. El-Dahr, J. Larraín, T. Obara, M. Oelgeschläger and E. Pera, aswell as all members of the laboratory, for critically reviewing the manuscriptand for helpful discussions; Dr E. Jones for the 4A6 and 3G8 antibodies; andDrs I. Dawid, R. Harland, R. Vignali, P. Vize and the NIBB/NIG/NBRP Xenopuslaevis EST project for plasmids. This work was supported by a grant fromNIH/NIDDK (#5R21DK077763-03) to O.W. Deposited in PMC for release after12 months.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/23/3927/DC1

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